The articles set forth in the Background of the Invention are each incorporated herein by reference.
Mammalian proteins present in clinical samples (e.g. whole blood, serum, plasma, cerebrospinal fluid, and urine) are useful as indicators of a disease state or a bodily condition. The amount and type of these proteins in the sample can provide a wealth of information to the clinician.
For example, the protein components of serum include albumin, alpha-1 lipoprotein, alpha-2 macroglobulin, beta-1 lipoprotein and immunoglobulins (including gammaglobulins). Albumin, the major protein of serum, is usually present in a concentration of between 4.0 and 5.0 g/dL. Decreased concentration of albumin can be indicative of renal disease; increased concentration of albumin is characteristic of dehydration. Elevated levels of alpha-1 lipoprotein can be indicative of chronic alcoholism or hyperestrogenism due to, e.g., pregnancy. Elevated levels of beta-1 lipoprotein can be indicative of increased cholesterol levels.
Mammalian proteins are charged proteins containing both cationic and anionic moieties. They thus lend themselves to analysis by capillary zone electrophoresis ("CZE"). CZE is a technique which permits rapid and efficient separations of charged substances. In general terms, CZE involves introduction of a sample into a capillary tube and the application of an electric field to the tube. The electric field pulls the sample through the tube and separates it into its constituent parts. I.e., each of the sample constituents has its own electrophoretic mobility; those having greater mobility travel through the capillary faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during the migration of the sample through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones. The detector measures the absorbance of light by each constituent at a specified wavelength; different constituents absorb light differently, and, because of this, the constituents can be differentiated from each other.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel, e.g. polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. In "open-tube" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon application of an electric field to the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution must flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in open CZE is as stable against conduction and diffusion as the gels utilized in gel CZE. Accordingly, separations can be obtained in open CZE quite similar to those obtained in gel-based electrophoresis.
Typically, the pH of the buffers utilized in open CZE are chosen with reference to the isoelectric points (pI) of the constituents in the sample. For example, the pI of serum albumin is 4.6; therefore, at pH 4.6, negatively charged and positively charged moieties of serum albumin are equal and the overall charge is neutral. However, as the pH is raised above the isoelectric point, the negatively charged moieties predominate and the net charge is negative. Thus, by selection of the proper pH, all of the species of the sample will be negatively charged. For serum samples, at pH greater than about 8.00, the majority of all serum-protein species will be negatively charged. Thus, manipulation of the isoelectric points of sample species can be used to ensure a proper charge distribution vis-a-vis the flow of such species through a charged capillary.
Typically, the results of CZE analysis are provided via an electropherogram, which depicts the discrete zones of the sample constituents as peaks of various height and width. Additionally, the results can be presented in terms of numerical data based upon the integrated area under each constituent peak.
From analysis of clinical samples, it is possible to determine a disease state or bodily condition by comparing the peaks obtained in an electropherogram of the sample with those obtained in an electropherogram of a known control. Thus, if a sample constituent peak from a clinical sample electropherogram is broader or more narrow or higher or not as high relative to the same sample constituent peak from the control electropherogram, it may be indicative of a disease state or a bodily condition.
A problem with CZE analysis of protein-containing samples, peptide-containing samples, and samples containing native and/or synthetic DNA and RNA, is that it is very difficult to accurately quantify the individual sample constituents. I.e, while it has been possible to visually compare relative peaks to determine if a potential disease condition exists, it has heretofore been a problem to accurately and consistently determine the precise quantitative amounts of the individual constituents of the sample.
Another problem encountered with capillary zone electrophoresis of such samples is that the constituents may appear on the electropherogram at different migration times with different samples. Stated again, a protein and/or peptide common to two different samples may show up at a different place on each of the electropherograms for such samples. This is due, in part, to the fact that the amount of time taken by each earlier sample constituent as it passes through the capillary will affect the migration time of latter sample constituents.
Previous attempts at quantitation of sample constituents have been reported. Cinnamic acid has been attempted as an internal standard for the determination of ferulic acid concentration in dog plasma after oral administration of .gamma.-oryzanol. Fujiwara, S. and Honda, S. "Determination of Cinnamic Acid and its Analogues by Electrophoresis in a Fused Silica Capillary Tube." Anal. Chem. 58:1811-1814 (1986). Phenol has been attempted as an internal standard for the determination of chlorinated phenol concentrations. Otsuka, K. et al. "Quantitation and Reproducibility in Chromatography with Micellar Solutions." J. of Chrom. 396:350-354 (1987). Additionally, coinjection of a known amount of a species to be analyzed is well documented.
Internal standards, i.e. standards that are detected within the constituent detection region, can lead to at least two problems. First, there is the potential for co-migration. The internal standard may migrate at or near a region where a sample constituent migrates leading to erroneous analysis of that constituent. This is because the sample constituent may appear to have a peak of greater height or width based upon its mixing with and the effect of the co-migrating standard. Second, because the amount of sample analyzed can affect its flow rate, the location of the internal standard peak on the electropherogram can vary depending on the amount of sample analyzed. If the peak location of the internal reference is artificially altered, quantitation of constituents based on such alterations would be inaccurate, incomprehensible or erroneous.
Accordingly, a need exists for an efficient and reliable method for rapidly quantifying protein-and/or peptide containing sample constituents using a capillary zone electrophoresis protocol.